Our activities
|
|
|
 |
 | |  | |
English spoken
Monday to Friday from 9h to 18h
| |  | |  |
|
Molecular Biology > Competent cells
 |
Bacteriology & competent cells
|
 |
|
^
|
|
|
OverExpress™ Strains competent cells
|
Avidis markets the innovative OverExpress™ C41(DE3) and C43(DE3) Escherichia coli strains (Miroux and Walker, 1996, J. Mol. Biol. 260, 289-298).
Starting from the classic BL21(DE3) strain, the new strains have been evolved to overexpress numerous toxic proteins that were difficult or impossible to produce before in bacteria.
The pAVD10 vector can be used to verify the identity of the strains BL21(DE3), C41(DE3) and C43(DE3).
The effectiveness of the OverExpress™ strains are proved by our customers: many pharmaceutical companies and hundred of public laboratories all over the world.
|
How to use Competent Cells
|
Transformation of OverExpressTM strains C41(DE3) and C43(DE3)
To maximise protein expression, fresh transformants are strongly recommended !
- Thaw an aliquot of CaCl2 competent cells on ice (for up to 15 minutes).
- Add 10-50 ng of plasmid DNA vector containing the gene of interest to a 50 µl aliquot of the cells.
- Mix by flipping, gently.
- Incubate the tube on ice for about 20 minutes.
- Place the tube at 42°C for 2 minutes, then put it back on ice for 3 minutes.
- Add 400 µl of LB medium and incubate the tube for between 15 minutes at 37°C.
- Spread a drop (up to 100µl) of bacteria on LB-agar Petri plates containing the appropriate antibiotic.
- Incubate the Petri plates at 37°C for 20±4 hours.
|
How to check the identity of the strains C41(DE3) and C43(DE3)
You may wish to verify the identity of the strains you are using; the pAVD10 vector is used for this purpose.
The procedure is as follows:.
On Day 1 :
- Thaw an aliquot of competent cells on ice for 10 minutes
- Add to the competent cells 1µl of pAVD10 plasmid (5ng/µl) on ice for 20 minutes .
- Heat-shock the transformation reaction in a 42°C water bath for 2 minutes.
- Incubate the reactions on ice for 3 minutes.
- Add 450 µl of LB medium to each transformation, and incubate the reactions at 37°C for 1 hour, with shaking, at 200 rpm.
- Plate:
- Incubate the plates overnight at 37°C.
On Day 2 :
Count the number of colonies on the LB-ampicillin and LB-ampicillin-IPTG plates.
Expected Results :
| |
BL21(DE3) |
C41(DE3) |
C43(DE3) |
| LB-Amp |
No colonies |
Colonies |
Colonies |
| LB-Amp-IPTG |
No colonies |
No colonies |
Colonies |
|
|
